Methods for genotyping polymorphisms in humans

ABSTRACT

The invention provides nucleic acid sequences that are complementary, in one embodiment, to a collection of human single nucleotide polymorphisms. The invention provides the sequences in such a way as to make them available for a variety of analyses including genotyping a large number of SNPs in parallel by, for example, allele specific hybridization. The invention also provides a collection of human SNPs that is useful for genetic analysis within and across populations. As such, the invention relates to diverse fields impacted by the nature of genetics, including biology, medicine, and medical diagnostics.

RELATED APPLICTIONS

The present application claims priority to U.S. Provisional Application Nos. 60/417,190 filed Oct. 8, 2002 and 60/470,475 filed May 14, 2003 and is a continuation-in-part of U.S. patent application Ser. No. 10/681,773 filed Oct. 7, 2003, the disclosures of which are each incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention provides pools of nucleic acid sequences and arrays of nucleic acid sequences that are useful for genotyping polymorphisms in nucleic acid samples derived from humans. The invention also provides a collection of SNPs that may be amplified reproducibly and genotyped in parallel using a single assay. The invention relates to diverse fields, including genetics, genomics, biology, population biology, medicine, and medical diagnostics.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on compact disk is hereby incorporated by reference. The file on the disk is named 3522.3seqlist.txt, the file is 1.45 MB and the date of creation is Jul. 12, 2004.

BACKGROUND

The past years have seen a dynamic change in the ability of science to comprehend vast amounts of data. Pioneering technologies such as nucleic acid arrays allow scientists to delve into the world of genetics in far greater detail than ever before. Exploration of genomic DNA has long been a dream of the scientific community. Held within the complex structures of genomic DNA lies the potential to identify, diagnose, or treat diseases like cancer, Alzheimer disease or alcoholism. Exploitation of genomic information from plants and animals may also provide answers to the world's food distribution problems.

Recent efforts in the scientific community, such as the publication of the draft sequence of the human genome in February 2001, have changed the dream of genome exploration into a reality. Genome-wide assays, however, must contend with the complexity of genomes; the human genome for example is estimated to have a complexity of 3×10⁹ base pairs. Novel methods of sample preparation and sample analysis that reduce complexity may provide for the fast and cost effective exploration of complex samples of nucleic acids, particularly genomic DNA.

Single nucleotide polymorphisms (SNPs) have emerged as the marker of choice for genome wide association studies and genetic linkage studies. Building SNP maps of the genome will provide the framework for new studies to identify the underlying genetic basis of complex diseases such as cancer, mental illness and diabetes. Due to the wide ranging applications of SNPs there is still a need for the development of robust, flexible, cost-effective technology platforms that allow for scoring genotypes in large numbers of samples.

SUMMARY OF THE INVENTION

The invention provides nucleic acid sequences that are complementary to particular regions of the human genome that are each known to contain a single nucleotide polymorphism. The invention further provides a collection of oligonucleotide probes that are useful for genotyping a selected set of SNPs found in the human genome. For example, in one embodiment the invention comprises an array comprising any 10 or more, 100 or more, 1000 or more, 10,000 or more, 100,000 or more, or 1,000,000 or more nucleic acid probes containing 15 or more consecutive nucleotides from the sequences listed in SEQ ID NOS: 1-10,204 or the complements thereof. Also contemplated are perfect match and mismatch probes that are subsequences of the sequences listed in SEQ ID Nos. 1-10,204 or the complements thereof. In a preferred embodiment the array comprises oligonucleotide probes that are 25 consecutive nucleotides from each of the sequences listed in SEQ ID NOS 1-10,204, each probe being a different 25 nucleotide sequence and each probe being attached to a solid support in a determinable position.

In a further embodiment, the invention comprises the use of any of the above arrays or fragments disclosed in SEQ ID NOS 1-10,204 to: monitor loss of heterozygosity; determine copy number, identify imprinted genes; genotype polymorphisms; determine allele frequencies in a population, characterize biallelic markers; produce genetic maps; detect linkage disequilibrium, determine allele frequencies, do association studies, analyze genetic variation, to identify markers linked to a phenotype or, compare genotypes between different individuals or populations. In a further embodiment the invention comprises a method of analysis comprising hybridizing one or more pools of nucleic acids to two or more probes, wherein the probes comprise at least 20 consecutive nucleotides from one of the sequences disclosed in SEQ ID NOS 1-10,204 and detecting said hybridization. In a further embodiment the invention comprises the use of any one or more 20 consecutive bases from one of the sequences disclosed in SEQ ID NOS 1-10,204 as a primer for PCR. Kits comprising collections of oligonucleotide probes are also disclosed. The oligonucleotide probes are perfectly complementary to one or the other allele of a selected SNP.

DETAILED DESCRIPTION OF THE INVENTION

a) General

The present invention has many preferred embodiments and relies on many patents, applications and other references for details known to those of the art. Therefore, when a patent, application, or other reference is cited or repeated below, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited.

As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

An individual is not limited to a human being but may also be other organisms including but not limited to mammals, plants, bacteria, or cells derived from any of the above.

Throughout this disclosure, various aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligátion, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, N.Y., Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

The present invention can employ solid substrates, including arrays in some preferred embodiments. Methods and techniques applicable to polymer (including protein) array synthesis have been described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730 (International Publication No. WO 99/36760) and PCT/US01/04285 (International Publication No. WO 01/58593), which are all incorporated herein by reference in their entirety for all purposes.

Genotyping methods are disclosed in, for example, U.S. Pat. Nos. 5,925,525, 6,268,141, 6,267,152, 6,525,185, 6,632,611, 6,673,579, 6,361,947 and 6,300,063.

Patents that describe synthesis techniques in specific embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098. Nucleic acid arrays are described in many of the above patents, but the same techniques are applied to polypeptide arrays.

Nucleic acid arrays that are useful in the present invention include those that are commercially available from Affymetrix (Santa Clara, Calif.) under the brand name GeneChip®. Example arrays are shown on the website at affymetrix.com.

The present invention also contemplates many uses for polymers attached to solid substrates. These uses include gene expression monitoring, profiling, library screening, genotyping and diagnostics. Gene expression monitoring and profiling methods can be shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. Nos. 10/442,021, 10/013,598 (U.S. patent application Publication 20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses are embodied in U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

The present invention also contemplates sample preparation methods in certain preferred embodiments. Prior to or concurrent with genotyping, the genomic sample may be amplified by a variety of mechanisms, some of which may employ PCR. See, for example, PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675, and each of which is incorporated herein by reference in their entireties for all purposes. The sample may be amplified on the array. See, for example, U.S. Pat. No. 6,300,070 and U.S. Ser. No. 09/513,300, which are incorporated herein by reference.

Other suitable amplification methods include the ligase chain reaction (LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317, each of which is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing the complexity of a nucleic sample are described in Dong et al., Genome Research 11, 1418 (2001), in U.S. Pat. No. 6,361,947, 6,391,592 and U.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. patent application Publication 20030096235), Ser. No. 09/910,292 (U.S. patent application Publication 20030082543), and Ser. No. 10/013,598.

Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. Cold Spring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983). Methods and apparatus for carrying out repeated and controlled hybridization reactions have been described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which are incorporated herein by reference

The present invention also contemplates signal detection of hybridization between ligands in certain preferred embodiments. See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensity data are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. Nos. 10/389,194, 60/493,495 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.

The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products of the invention typically include computer readable medium having computer-executable instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. The computer executable instructions may be written in a suitable computer language or combination of several languages. Basic computational biology methods are described in, for example Setubal and Meidanis et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics: Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S. Pat. No. 6,420,108.

The present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.

Additionally, the present invention may have preferred embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559 (U.S. Publication No. 20020183936), Ser. No. 10/065,856, 10/065,868, 10/328,818, 10/328,872, 10/423,403, and 60/482,389.

b) Definitions

The term “admixture” refers to the phenomenon of gene flow between populations resulting from migration. Admixture can create linkage disequilibrium (LD).

The term “allele” as used herein is any one of a number of alternative forms a given locus (position) on a chromosome. An allele may be used to indicate one form of a polymorphism, for example, a biallelic SNP may have possible alleles A and B. An allele may also be used to indicate a particular combination of alleles of two or more SNPs in a given gene or chromosomal segment. The frequency of an allele in a population is the number of times that specific allele appears divided by the total number of alleles of that locus.

The term “array” as used herein refers to an intentionally created collection of molecules which can be prepared either synthetically or biosynthetically. The molecules in the array can be identical or different from each other. The array can assume a variety of formats,for example, libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports.

The term “biomonomer” as used herein refers to a single unit of biopolymer, which can be linked with the same or other biomonomers to form a biopolymer (for example, a single amino acid or nucleotide with two linking groups one or both of which may have removable protecting groups) or a single unit which is not part of a biopolymer. Thus, for example, a nucleotide is a biomonomer within an oligonucleotide biopolymer, and an amino acid is a biomonomer within a protein or peptide biopolymer; avidin, biotin, antibodies, antibody fragments, etc., for example, are also biomonomers.

The term “biopolymer” or sometimes refer by “biological polymer” as used herein is intended to mean repeating units of biological or chemical moieties. Representative biopolymers include, but are not limited to, nucleic acids, oligonucleotides, amino acids, proteins, peptides, hormones, oligosaccharides, lipids, glycolipids, lipopolysaccharides, phospholipids, synthetic analogues of the foregoing, including, but not limited to, inverted nucleotides, peptide nucleic acids, Meta-DNA, and combinations of the above.

The term “biopolymer synthesis” as used herein is intended to encompass the synthetic production, both organic and inorganic, of a biopolymer. Related to a bioploymer is a “biomonomer”.

The term “combinatorial synthesis strategy” as used herein refers to a combinatorial synthesis strategy is an ordered strategy for parallel synthesis of diverse polymer sequences by sequential addition of reagents which may be represented by a reactant matrix and a switch matrix, the product of which is a product matrix. A reactant matrix is a 1 column by m row matrix of the building blocks to be added. The switch matrix is all or a subset of the binary numbers, preferably ordered, between 1 and m arranged in columns. A “binary strategy” is one in which at least two successive steps illuminate a portion, often half, of a region of interest on the substrate. In a binary synthesis strategy, all possible compounds which can be formed from an ordered set of reactants are formed. In most preferred embodiments, binary synthesis refers to a synthesis strategy which also factors a previous addition step. For example, a strategy in which a switch matrix for a masking strategy halves regions that were previously illuminated, illuminating about half of the previously illuminated region and protecting the remaining half (while also protecting about half of previously protected regions and illuminating about half of previously protected regions). It will be recognized that binary rounds may be interspersed with non-binary rounds and that only a portion of a substrate may be subjected to a binary scheme. A combinatorial “masking” strategy is a synthesis which uses light or other spatially selective deprotecting or activating agents to remove protecting groups from materials for addition of other materials such as amino acids.

The term “complementary” as used herein refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Alternatively, complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90% complementary. See, M. Kanehisa Nucleic Acids Res. 12:203 (1984), incorporated herein by reference.

The term “effective amount” as used herein refers to an amount sufficient to induce a desired result.

The term “genome” as used herein is all the genetic material in the chromosomes of an organism. DNA derived from the genetic material in the chromosomes of a particular organism is genomic DNA. A genomic library is a collection of clones made from a set of randomly generated overlapping DNA fragments representing the entire genome of an organism.

The term “genotype” as used herein refers to the genetic information an individual carries at one or more positions in the genome. A genotype may refer to the information present at a single polymorphism, for example, a single SNP. For example, if a SNP is biallelic and can be either an A or a C then if an individual is homozygous for A at that position the genotype of the SNP is homozygous A or AA. Genotype may also refer to the information present at a plurality of polymorphic positions.

The term “Hardy-Weinberg equilibrium” (HWE) as used herein refers to the principle that an allele that when homozygous leads to a disorder that prevents the individual from reproducing does not disappear from the population but remains present in a population in the undetectable heterozygous state at a constant allele frequency.

The term “hybridization” as used herein refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide; triple-stranded hybridization is also theoretically possible. The resulting (usually) double-stranded polynucleotide is a “hybrid.” The proportion of the population of polynucleotides that forms stable hybrids is referred to herein as the “degree of hybridization.” Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than about 1 M and a temperature of at least 25° C. Examples of hybridization conditions include: 5× SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. are suitable for allele-specific probe hybridizations or conditions of 100 mM MES, 1 M [Na⁺], 20 mM EDTA, 0.01% Tween-20 and a temperature of 30-50° C., preferably at about 45-50° C. Hybridizations may be performed in the presence of agents such as herring sperm DNA at about 0.1 mg/ml, acetylated BSA at about 0.5 mg/ml. In a preferred embodiment 70 ul of labeled DNA is mixed with 190 ul of the following hybridization cocktail: 0.056 M MES, 5.0% DMSO, 2.50× Denhardt's Solution, 5.77 mM EDTA, 0.115 mg/mL Herring Sperm DNA (10 mg/mL), 11.5 μg/mL Human Cot-1, 0.0115% Tween-20, and 2.69 M (3%) TMACL and hybridized to a genotyping array at 16° C.

As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, the combination of parameters is more important than the absolute measure of any one alone. Hybridization conditions suitable for microarrays are described in the Gene Expression Technical Manual, 2004 and the GeneChip Mapping Assay Manual, 2004.

The term “hybridization probes” as used herein are oligonucleotides capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include oligonucleotides, peptide nucleic acids, as described in Nielsen et al., Science 254, 1497-1500 (1991), LNAs, as described in Koshkin et al. Tetrahedron 54:3607-3630, 1998, and U.S. Pat. No. 6,268,490 and other nucleic acid analogs and nucleic acid mimetics.

The term “hybridizing specifically to” as used herein refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence or sequences under stringent conditions when that sequence is present in a complex mixture (for example, total cellular) DNA or RNA.

The term “initiation biomonomer” or “initiator biomonomer” as used herein is meant to indicate the first biomonomer which is covalently attached via reactive nucleophiles to the surface of the polymer, or the first biomonomer which is attached to a linker or spacer arm attached to the polymer, the linker or spacer arm being attached to the polymer via reactive nucleophiles.

The term “isolated nucleic acid” as used herein mean an object species invention that is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition). Preferably, an isolated nucleic acid comprises at least about 50, 80 or 90% (on a molar basis) of all macromolecular species present. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods).

The term “ligand” as used herein refers to a molecule that is recognized by a particular receptor. The agent bound by or reacting with a receptor is called a “ligand,” a term which is definitionally meaningful only in terms of its counterpart receptor. The term “ligand” does not imply any particular molecular size or other structural or compositional feature other than that the substance in question is capable of binding or otherwise interacting with the receptor. Also, a ligand may serve either as the natural ligand to which the receptor binds, or as a functional analogue that may act as an agonist or antagonist. Examples of ligands that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (for example, opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, substrate analogs, transition state analogs, cofactors, drugs, proteins, and antibodies.

The term “linkage analysis” as used herein refers to a method of genetic analysis in which data are collected from affected families, and regions of the genome are identified that co-segregated with the disease in many independent families or over many generations of an extended pedigree. A disease locus may be identified because it lies in a region of the genome that is shared by all affected members of a pedigree.

The term “linkage disequilibrium” or sometimes referred to as “allelic association” as used herein refers to the preferential association of a particular allele or genetic marker with a specific allele, or genetic marker at a nearby chromosomal location more frequently than expected by chance for any particular allele frequency in the population. For example, if locus X has alleles A and B, which occur equally frequently, and linked locus Y has alleles C and D, which occur equally frequently, one would expect the combination AC to occur with a frequency of 0.25. If AC occurs more frequently, then alleles A and C are in linkage disequilibrium. Linkage disequilibrium may result from natural selection of certain combination of alleles or because an allele has been introduced into a population too recently to have reached equilibrium with linked alleles. The genetic interval around a disease locus may be narrowed by detecting disequilibrium between nearby markers and the disease locus. For additional information on linkage disequilibrium see Ardlie et al., Nat. Rev. Gen. 3:299-309, 2002.

The term “lod score” or “LOD” is the log of the odds ratio of the probability of the data occurring under the specific hypothesis relative to the null hypothesis. LOD=log [probability assuming linkage/probability assuming no linkage].

The term “mixed population” or sometimes refer by “complex population” as used herein refers to any sample containing both desired and undesired nucleic acids. As a non-limiting example, a complex population of nucleic acids may be total genomic DNA, total genomic RNA or a combination thereof. Moreover, a complex population of nucleic acids may have been enriched for a given population but include other undesirable populations. For example, a complex population of nucleic acids may be a sample which has been enriched for desired messenger RNA (mRNA) sequences but still includes some undesired ribosomal RNA sequences (rRNA).

The term “monomer” as used herein refers to any member of the set of molecules that can be joined together to form an oligomer or polymer. The set of monomers useful in the present invention includes, but is not restricted to, for the example of (poly)peptide synthesis, the set of L-amino acids, D-amino acids, or synthetic amino acids. As used herein, “monomer” refers to any member of a basis set for synthesis of an oligomer. For example, dimers of L-amino acids form a basis set of 400 “monomers” for synthesis of polypeptides. Different basis sets of monomers may be used at successive steps in the synthesis of a polymer. The term “monomer” also refers to a chemical subunit that can be combined with a different chemical subunit to form a compound larger than either subunit alone.

The term “mRNA” or sometimes refer by “mRNA transcripts” as used herein, include, but not limited to pre-mRNA transcript(s), transcript processing intermediates, mature mRNA(s) ready for translation and transcripts of the gene or genes, or nucleic acids derived from the mRNA transcript(s). Transcript processing may include splicing, editing and degradation. As used herein, a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template. Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc., are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample. Thus, mRNA derived samples include, but are not limited to, mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.

The term “nucleic acid library” or sometimes refer by “array” as used herein refers to an intentionally created collection of nucleic acids which can be prepared either synthetically or biosynthetically and screened for biological activity in a variety of different formats (for example, libraries of soluble molecules; and libraries of oligos tethered to resin beads, silica chips, or other solid supports). Additionally, the term “array” is meant to include those libraries of nucleic acids which can be prepared by spotting nucleic acids of essentially any length (for example, from 1 to about 1000 nucleotide monomers in length) onto a substrate. The term “nucleic acid” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Thus the terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include analogs such as those described herein. These analogs are those molecules having some structural features in common with a naturally occurring nucleoside or nucleotide such that when incorporated into a nucleic acid or oligonucleoside sequence, they allow hybridization with a naturally occurring nucleic acid sequence in solution. Typically, these analogs are derived from naturally occurring nucleosides and nucleotides by replacing and/or modifying the base, the ribose or the phosphodiester moiety. The changes can be tailor made to stabilize or destabilize hybrid formation or enhance the specificity of hybridization with a complementary nucleic acid sequence as desired.

The term “nucleic acids” as used herein may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. See Albert L. Lehninger, PRINCIPLES OF BIOCHEMISTRY, at 793-800 (Worth Pub. 1982). Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glucosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally-occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

The term “oligonucleotide” or sometimes refer by “polynucleotide” as used herein refers to a nucleic acid ranging from at least 2, preferable at least 8, and more preferably at least 20 nucleotides in length or a compound that specifically hybridizes to a polynucleotide. Polynucleotides of the present invention include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) which may be isolated from natural sources, recombinantly produced or artificially synthesized and mimetics thereof. A further example of a polynucleotide of the present invention may be peptide nucleic acid (PNA). The invention also encompasses situations in which there is a nontraditional base pairing such as Hoogsteen base pairing which has been identified in certain tRNA molecules and postulated to exist in a triple helix. “Polynucleotide” and “oligonucleotide” are used interchangeably in this application.

The term “polymorphism” as used herein refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymorphism may comprise one or more base changes, an insertion, a repeat, or a deletion. A polymorphic locus may be as small as one base pair. Polymorphic markers include restriction fragment length polymorphisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu. The first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms. A diallelic polymorphism has two forms. A triallelic polymorphism has three forms. Single nucleotide polymorphisms (SNPs) are included in polymorphisms.

The term “primer” as used herein refers to a single-stranded oligonucleotide capable of acting as a point of initiation for template-directed DNA synthesis under suitable conditions for example, buffer and temperature, in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase. The length of the primer, in any given case, depends on, for example, the intended use of the primer, and generally ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with such template. The primer site is the area of the template to which a primer hybridizes. The primer pair is a set of primers including a 5′ upstream primer that hybridizes with the 5′ end of the sequence to be amplified and a 3′ downstream primer that hybridizes with the complement of the 3′ end of the sequence to be amplified.

The term “probe” as used herein refers to a surface-immobilized molecule that can be recognized by a particular target. See U.S. Pat. No. 6,582,908 for an example of arrays having all possible combinations of probes with 10, 12, and more bases. Examples of probes that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (for example, opioid peptides, steroids, etc.), hormone receptors, peptides; enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

The term “receptor” as used herein refers to a molecule that has an affinity for a given ligand. Receptors may be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of receptors which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Receptors are sometimes referred to in the art as anti-ligands. As the term receptors is used herein, no difference in meaning is intended. A “Ligand Receptor Pair” is formed when two macromolecules have combined through molecular recognition to form a complex. Other examples of receptors which can be investigated by this invention include but are not restricted to those molecules shown in U.S. Pat. No. 5,143,854, which is hereby incorporated by reference in its entirety.

The term “solid support”, “support”, and “substrate” as used herein are used interchangeably and refer to a material or group of materials having a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) will take the form of beads, resins, gels, microspheres, or other geometric configurations. See U.S. Pat. No. 5,744,305 for exemplary substrates.

The term “target” as used herein refers to a molecule that has an affinity for a given probe. Targets may be naturally-occurring or man-made molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, oligonucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Targets are sometimes referred to in the art as anti-probes. As the term targets is used herein, no difference in meaning is intended. A “Probe Target Pair” is formed when two macromolecules have combined through molecular recognition to form a complex.

C. Interrogation of Selected Human SNPs

SEQ ID NOS 1-10,204, are disclosed. The sequences provided correspond to one strand of the genomic DNA including a SNP position and 16 bases 5′ of the SNP position and 16 bases 3′ of the SNP position. The polymorphic base is at position 17 of the sequence in the sequence listing. For example, SEQ ID No. 1 is 5′-agctgagcag aaagagKttg ggtcttagtg tct-3′ and the SNP position is represented by K. The symbols Y,K, M and R are used to represent the SNP position where Y is T or C, K is G or T, M is A or C, R is G or A, W is A or T and S is G or C. So, for SEQ ID NO 1 the SNP has alleles G and T. Each of SEQ ID Nos. 1-10,204 represents a single SNP from the human genome and 32 bases of sequence flanking each SNP. The sequences also represent a set of probes to genotype the corresponding SNP by allele specific hybridization. In a preferred embodiment the probes comprise 20 to 25 contiguous nucleotides from one of SEQ ID Nos. 1-10,204. SNPs were selected to be included in the list of SNPs and to be interrogated by the mapping array from public databases of SNPs such as dbSNP, The SNP Consortium (TSC) and from SNPs identified by Perlegen Sciences, Inc. SNPs were selected through a screening and validation process that evaluated accuracy of genotyping calls, rate of genotyping calls, and physical distribution in the genome.

The present invention includes: the sequences listed in SEQ ID Nos. 1-10,204 and probes comprising 11 to 33 contiguous bases of any of the sequences in SEQ ID Nos. 1-10,204 or the complement thereof. In a preferred embodiment the probes comprise 20-25 contiguous bases of a sequence from SEQ ID Nos. 1-10,204 or the complement thereof. Also contemplated are corresponding mismatch probes that include a single base mismatch, preferably at a central position of the probe, and longer nucleotide sequences that comprise a contiguous subsequence of at least 15 bases of one of the sequences listed in SEQ ID Nos. 1-10,204.

The set of SNPs represented by SEQ ID Nos. 1-10,204 varies from the set disclosed in U.S. patent application Ser. No. 10/681,773 by removal of approximately 1400 of the SNPs disclosed in Ser. No. 10/681,773 and addition of 68 new SNPs, corresponding to SEQ ID Nos. 10,137-10,204. The 1400 SNPs were removed because they have lower call rates than the remaining SNPs or are close to another SNP in the set. The reduced number of probes required to genotype the reduced number of SNPs allows the probes of the array to be tiled in 8 μm² features on a 169 format array with array size about 7 μm² with more than 410,000 probes. The 68 new SNPs were selected to provide better SNP coverage on chromosomes 19 and the X chromosome. The probes of the array are allele specific so if the SNP is either T or a C then there is a probe or probe set that is complementary to the T allele and a probe or probe set that is complementary to the C allele and the probe for the T allele is in a different feature of the array than the probe for the C allele.

In a preferred embodiment the array comprises sets of probes to detect the SNPs represented by SEQ ID NOS 1-10,204 or a subset of those SNPs. Arrays may detect at least 1,000, 5,000, or 10,000 of the selected SNPs. In one embodiment there are 40 probes for each SNP, comprising 10 quartets of 4 probes. Each quartet consists of a perfect match probe for allele A, a perfect match probe for allele B, a mismatch probe for allele A and a mismatch probe for allele B. The mismatch probe is identical to the perfect match probe with the exception that the central base in the mismatch probe, position 13 in a 25 nucleotide probe, is the complement of the perfect match base. For example, if the perfect match probe has a G at position 13 the mismatch probe has a C at that position. One quartet varies from another in the position of the polymorphic base, for example in one quartet the polymorphic base may be at the central or 0 position of the probe, which is the 13^(th) nucleotide from the 5′ end of a 25 nucleotide probe. In the other quartets the polymorphic base may be shifted 5′ or 3′ of the 0 position. Thus, each 33 nucleotide sequence in the sequence listing represents at least 40 different probes that contain at least 15 contiguous nucleotides of a sequence from the sequence listing. Preferably the probes are 25 contiguous nucleotides of a sequence from the sequence listing. A probe set that is exemplary of a probe set of a preferred genotyping array is shown in Table 1. TABLE 1 SEQ ID NO PM/MM, A/G Position Probe Sequence Strand 10,205 PM A −4 tcatttaacattTgtgTtaacaaaa 0 10,206 MM A −4 tcatttaacattAgtgTtaacaaaa 0 10,207 PM A −2 atttaacatttgTgTtaacaaaaac 0 10,208 MM A −2 atttaacatttgAgTtaacaaaaac 0 10,209 PM A −1 tttaacatttgtGTtaacaaaaact 0 10,210 MM A −1 tttaacatttgtCTtaacaaaaact 0 10,211 PM A 0 ttaacatttgtgTtaacaaaaactc 0 10,212 MM A 0 ttaacatttgtgAtaacaaaaactc 0 10,213 PM A 1 taacatttgtgTTaacaaaaactcc 0 10,214 MM A 1 taacatttgtgTAaacaaaaactcc 0 10,215 PM A 4 catttgtgTtaaCaaaaactcctcc 0 10,216 MM A 4 catttgtgTtaaGaaaaactcctcc 0 10,217 PM G −4 tcatttaacattTgtgCtaacaaaa 0 10,218 MM G −4 tcatttaacattAgtgCtaacaaaa 0 10,219 PM G −2 atttaacatttgTgCtaacaaaaac 0 10,220 MM G −2 atttaacatttgAgCtaacaaaaac 0 10,221 PM G −1 tttaacatttgtGCtaacaaaaact 0 10,222 MM G −1 tttaacatttgtCCtaacaaaaact 0 10,223 PM G 0 ttaacatttgtgCtaacaaaaactc 0 10,224 MM G 0 ttaacatttgtgGtaacaaaaactc 0 10,225 PM G 1 taacatttgtgCTaacaaaaactcc 0 10,226 MM G 1 taacatttgtgCAaacaaaaactcc 0 10,227 PM G 4 catttgtgCtaaCaaaaactcctcc 0 10,228 MM G 4 catttgtgCtaaGaaaaactcctcc 0 10,229 PM A −4 ttttgttaAcacAaatgttaaatga 1 10,230 MM A −4 ttttgttaAcacTaatgttaaatga 1 10,231 PM A −2 gtttttgttaAcAcaaatgttaaat 1 10,232 MM A −2 gtttttgttaAcTcaaatgttaaat 1 10,233 PM A −1 agtttttgttaACacaaatgttaaa 1 10,234 MM A −1 agtttttgttaAGacaaatgttaaa 1 10,235 PM A 0 gagtttttgttaAcacaaatgttaa 1 10,236 MM A 0 gagtttttgttaTcacaaatgttaa 1 10,237 PM G −4 ttttgttaGcacAaatgttaaatga 1 10,238 MM G −4 ttttgttaGcacTaatgttaaatga 1 10,239 PM G −2 gtttttgttaGcAcaaatgttaaat 1 10,240 MM G −2 gtttttgttaCcTcaaatgttaaat 1 10,241 PM G −1 agtttttgttaGCacaaatgttaaa 1 10,242 MM G −1 agtttttgttaGGacaaatgttaaa 1 10,243 PM G 0 gagtttttgttaGcacaaatgttaa 1 10,244 MM G 0 gagtttttgttaCcacaaatgttaa 1

The exemplary probe set shown in Table 1 has 40 probes. There are 10 sets of 4 probes. Each set has 2 perfect match probes, 1 for each allele (PM A and PM G) and 2 mismatch probes (MM A and MM G). In this set there are 24 probes, (6 probe sets), for the 0 strand and 16 probes, (4 probe sets), for the 1 strand. The probe sets for the 0 strand correspond to the SNP allele at positions −4, −2, −1, 0, 1 and 4 and the probe sets for the 1 strand correspond to the SNP allele at positions −4, −2, −1 and 0. As shown by this example there can be an unequal number of probe sets for the 0 and 1 strand. PM A indicates perfect match probes for allele A and PM G indicates perfect match probes for allele G. Mismatch probes for allele A and G are indicated by MM A and MM G, respectively. The strands are indicated by 0 or 1, where 0 and 1 are complementary strands of a double stranded DNA.

In a preferred embodiment the optimal 10 quartets are selected for each SNP. This may be 5 quartets for each of the two strands or an unequal number of quartets from each strand, for example 6 from one strand and 4 for the other as in the example in Table 1. There may be 7 and 3, 8 and 2, 9 and 1 or 10 and 0. For example, the 5 quartets may be 0, +2, +4, −3, and −4 on one strand and 0, +1, +3, −2, and −4 on the opposite strand. In some embodiments the quartets for one strand correspond to the quartets from the other strand so that the probes from one quartet are the complements of the probes from the corresponding quartet.

The central position, which varies between PM and MM, and the allele specific position, which varies between PM A and PM G, are capitalized in Table 1. For example, Table 1 SEQ ID No. 10,205 has a T at position 13 and a T at position 17 and is the PM A probe. SEQ ID No. 10,206 has an A at position 13 and a T at position 17. Position 13 is the position of the mismatch base and position 17 is the position of the allele specific SNP base. The SNP position is shifted to the −4 position relative to the 0 strand. The −4 G allele probes are SEQ ID Nos. 10,217 and 10,218 and they have a C at position 17. The −4 probes for the A allele for the 1 strand, the opposite strand, are SEQ ID Nos. 10,229 and 10,230. The mismatch is at position 13 and the SNP allele is at position 9.

Accordingly, for each nucleic acid sequence listed in SEQ ID NOS 1-10,204, this disclosure includes a probe comprising any contiguous length of from 15 to 33 nucleotides, preferably between 20 and 25 nucleotides, from a sequence in the list or the complement of a sequence in the list. The oligonucleotides may also comprise any contiguous length from 15 to 33 nucleotides from a sequence in the list or the complement of a sequence in the list including a single base mismatch. The position of the mismatch is preferably located at the central position of the probe, for example, for a probe of 25 nucleotides, the mismatch position would be position 13. In another embodiment the mismatch position may be located anywhere in the nucleic acid sequence and may comprise one or more bases. Generally the sequences correspond to SNPs represented by SEQ ID Nos. 1-10,204 which include the SNP position and sequences surrounding the SNP. The SNPs are preferably biallelic but may be triallelic and the probes in a preferred embodiment are used to distinguish between different alleles of a SNP. Frequency of SNPs vary between populations so a SNP that is biallelic in one population may not be polymorphic in another population or may be represented by different alleles.

The present invention includes: the sequences listed in SEQ ID NOS 1-10,204 and the complement of these sequences as well as mismatch probes, longer nucleotide sequences which include the nucleic acid sequences listed in SEQ ID NOS 1-10,204 and the complement of these sequences and sub-sequences greater than 9 nucleotides in length of the target nucleic acid sequences listed in SEQ ID NOS 1-10,204 and the complement of these sequences. Also disclosed are oligonucleotides that comprise 15 to 33 contiguous nucleotides from one of the sequences in SEQ ID No. 1-10,204 or the complement of SEQ ID No. 1-10,204 and additional sequence 5′ or 3′. For example, oligonucleotides or probes may comprise one or more tag sequences, universal priming sequence, or restriction enzyme recognition sequences upstream or downstream of a region comprising 15 to 33 contiguous nucleotides of SEQ ID No. 1-10,204. The oligonucleotides may be attached to a solid support, for example, each different sequence may be attached to one or more beads. In one embodiment an oligonucleotide that is complementary to each allele of each of the SNPs represented by SEQ ID No. 1-10,204 is attached to an array in a determinable location that is different from a plurality of other different sequence oligonucleotides. The oligonucleotides may be extended in an allele specific manner and extension detected. For example, extension may be with labeled nucleotides and may also be allele specific. Differently tagged oligos may be used for each allele of each SNP. See, for example, U.S. Pat. Nos. 6,709,816, 6,287,778, and 6,638,719.

SNPs were selected from the publicly available database of human SNPs. The selected SNPs are from the group of SNPs that are present on XbaI fragments of 250 to 2000 base pairs. A computer system was used to predict fragments that would result when the human genome is digested with XbaI. In some embodiments the present invention provides a pool of unique nucleotide sequences complementary to SNPs and sequence surrounding SNPs which alone, or in combinations of 2 or more, 10 or more, 100 or more, 1,000 or more, 10,000 or more , 100,000 or more or 400,000 or more, can be used for a variety of applications. In a preferred embodiment an array comprising allele specific probes for each allele of each SNP represented by SEQ ID Nos. 1-10,204 is disclosed. The probes are preferably present at a feature density of greater than 400,000 probes per cm2 and more preferably each different probe is present in a feature that is about 8 μm×8 μm or smaller.

In one embodiment, the present invention provides for a pool of unique nucleotide sequences which are complementary to Human SNPs and sequence surrounding SNPs formed into a high density array of probes suitable for array based massive parallel gene expression. Array based methods for SNP analysis and genotyping are disclosed and discussed in detail in U.S. Pat. Nos. 6,361,947 and 6,368,799 which are incorporated herein by reference for all purposes. Generally those methods of SNP analysis involve: (1) providing a pool of target nucleic acids comprising one or more target sequence(s), (2) amplifying a collection of target sequences, (3) hybridizing the amplified nucleic acid sample to a high density array of probes, and (4) detecting the hybridized nucleic acids and determining the presence or absence of one or more alleles for one or more SNPs.

The development of Very Large Scale Immobilized Polymer Synthesis or VLSIPS™ technology has provided methods for making very large arrays of nucleic acid probes in very small arrays. See U.S. Pat. No. 5,143,854 and PCT Nos. WO 90/15070 and 92/10092, and Fodor et al., Science, 251:767-77 (1991), each of which is incorporated herein by reference. U.S. Pat. Nos. 5,800,992 and 6,040,138 describe methods for making arrays of nucleic acid probes that can be used to detect the presence of a nucleic acid containing a specific nucleotide sequence. Methods of forming high-density arrays of nucleic acids, peptides and other polymer sequences with a minimal number of synthetic steps are known. The nucleic acid array can be synthesized on a solid substrate by a variety of methods, including, but not limited to, light-directed chemical coupling, and mechanically directed coupling.

In one embodiment probes are present on the array so that each SNP is represented by a collection of probes. The array may comprise between 4 and 80 probes for each SNP. In one embodiment the collection comprises, between 20 and 40 probes for each SNP. In another embodiment the array comprises about 40 to about 56 probes for each SNP. In a preferred embodiment the collection comprises about 40 probes for each SNP, 20 for each allele. In one embodiment the probes may be present in sets of 8 probes that correspond to a PM probe for each of two alleles, a MM probe for each of 2 alleles, and the corresponding probes for the opposite strand. In another embodiment the probes may be present in sets of 4 probes that correspond to a PM probe for each of two alleles and a MM probe for each of 2 alleles. For each allele there may be a perfect match, a perfect mismatch, an antisense match and an antisense mismatch probe. The polymorphic position may be the central position of the probe region, for example, the probe region may be 25 nucleotides and the polymorphic allele may be in the middle with 12 nucleotides on either side. In other probe sets the polymorphic position may be offset from the center. For example, the polymorphic position may be from 1 to 7 bases from the central position on either the 5′ or 3′ side of the probe. The interrogation position, which is changed in the mismatch probes, may remain at the center position. In one embodiment there are 56 probes for each SNP: the 8 probes corresponding to the polymorphic position at the center or 0 position and 8 probes for the polymorphic position at each of the following positions: −4, −2, −1, +1, +3 and +4 relative to the central or 0 position. In another embodiment 40 probes are used, 8 for the 0 position and 8 for each of 4 additional positions selected from: −4, −2, −1, +1, +3 and +4 relative to the central or 0 position. The probes sets used may vary depending on the SNP, for example, for one SNP the probes may be −4, −2, 0, +1 and +4 and for another SNP they may be −2, −1, 0, +1 and +4. Empirical data may be used to choose which probe sets to use on an array. In another embodiment 24 or 32 probes may be used for one or more SNPs.

In many embodiments pairs are present in perfect match and mismatch pairs, one probe in each pair being a perfect match to the target sequence and the other probe being identical to the perfect match probe except that the central base is a homo-mismatch. Mismatch probes provide a control for non-specific binding or cross-hybridization to a nucleic acid in the sample other than the target to which the probe is directed. Thus, mismatch probes may be used to indicate whether hybridization is or is not specific. For example, if the target is present, the perfect match probes should be consistently brighter than the mismatch probes because fluorescence intensity, or brightness, corresponds to binding affinity. ( See e.g., U.S. Pat. No. 5,324,633 and PCT No. WO 98/11223 which are incorporated herein for all purposes.

In another embodiment, the current invention may be combined with known methods to genotype polymorphism in a wide variety of contexts. For example, the methods may be used to do association studies, identify candidate genes associated with a phenotype, genotype SNPs in clinical populations, or correlate genotype information to clinical phenotypes. The SNPs of represented by SEQ ID Nos. 1-10,204 have been selected based on a number of criteria that make them suitable for complex genetic analysis, for example, linkage analysis and association studies. The SNPs are spaced throughout the genome at an average distance of 210 Kb from one another and they are known to be polymorphic in multiple populations. The panel of SNPs or a subset of these SNPs may be genotyped by any method available. See, Color Atlas of Genetics (2^(nd) ed), Ed. Passarge (2001) Thieme, NY, N.Y., which is incorporated by reference.

For a discussion of genotyping analysis methods see, for example, Elena and Lenski Nature Reviews, Genetics 4:457-469 (2003), Twyman and Primrose, Pharmacogenomics 4:67-79 (2003), Hirschhorn et al. Genetics in Medicine 4:45-61 (2002) and Glazier et al. Science 298:2345-2349 (2002) each of which is incorporated herein by references for all purposes. For a discussion of high throughput genotyping approaches see, for example, Jenkins and Gibson, Comp Funct Genom 2002; 3:57-66 which is incorporated herein by reference. For a review of methods of haplotype analysis in population genetics and association studies see, for example, Zhao et al. Pharmacogenomics 4:171-178 (2003), which is incorporated herein by reference.

One skilled in the art will appreciate that a wide range of applications will be available using 2 or more, 10 or more, 100 or more, 1000 or more, 10,000 or more, 100,000 or more, or more of the SEQ ID NOS 1-10,204 sequences as probes for polymorphism detection and analysis. The combination of the DNA array technology and the Human SNP specific probes in this disclosure is a powerful tool for genotyping and mapping disease loci.

In many embodiments the target sequences are a subset that is representative of a larger set. For example, the target sequences may be at least 1,000, 5,000, 10,000 or 100,000 to 10,000, 20,000, 100,000, 1,500,000 or 3,000,000 SNPs that may be representative of a larger population of SNPs present in a population of individuals. The target sequences may be dispersed throughout a genome, including for example, sequences from each chromosome, or each arm of each chromosome. Target sequences may be representative of haplotypes or particular phenotypes or collections of phenotypes. For a description of haplotypes see, for example, Gabriel et al., Science, 296:2225-9 (2002), Daly et al. Nat Genet., 29:229-32 (2001) and Rioux et al., Nat Genet., 29:223-8 (2001), each of which is incorporated herein by reference in its entirety.

In another embodiment, the present invention may be used for cross-species comparisons. One skilled in the art will appreciate that it is often useful to determine whether a SNP present in one species, for example human, is present in a conserved format in another species, including, without limitation, gorilla, chimp, mouse, rat, chicken, zebrafish, Drosophila, or yeast. See e.g. Andersson et al., Mamm. Genome, 7(10):717-734 (1996), which is hereby incorporated by reference for all purposes, which describes the utility of cross-species comparisons. The use of 2 or more, 10 or more, 100 or more, 1000 or more, 10,000 or more, 100,000 or more of the sequences disclosed in this invention in an array can be used to determine whether any sequence from one or more of the Human genes represented by the sequences disclosed in this invention is conserved in another species by, for example, hybridizing genomic nucleic acid samples from another species to an array comprised of the sequences disclosed in this invention.

In another embodiment of the invention, the sequences of this invention and arrays comprising probes comprising the disclosed sequences and fragments thereof, may be used to generate primers directed to their corresponding genes as disclosed in the GenBank, dbSNP or any other public database. The sequences provided in the sequence listing may be used to identify the region of the genome containing the associated SNP and the frequency of each allele in a given population. Primers may be used in such basic techniques as sequencing or PCR, see e.g., Sambrook, incorporated by reference above. In one embodiment PCR is allele specific.

In a preferred embodiment, the hybridized nucleic acids are detected by detecting one or more labels attached to the sample nucleic acids. The labels may be incorporated by any of a number of means well known to those of skill in the art. In one embodiment, the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acids. The label may be, for example, fluorescent or chemiluminescent. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a labeled amplification product. In another embodiment, transcription amplification using a labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to the original nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplification product after the amplification is completed. Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example, nick translation or end-labeling (e.g. with a labeled RNA) by kinasing the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore). In another embodiment label is added to the end of fragments using terminal deoxytransferase (TdT).

Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include, but are not limited to: biotin for staining with labeled streptavidin conjugate; anti-biotin antibodies, magnetic beads (e.g., Dynabeads™); fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like); radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P); phosphorescent labels; enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA); and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each of which is hereby incorporated by reference in its entirety for all purposes.

Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters; fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.

D. Methods of Use

The disclosed arrays, sequences and probes can be used for a wide variety of applications including, for example, linkage and association studies, identification of candidate gene regions, genotyping clinical populations, correlation of genotype information to phenotype information, loss of heterozygosity analysis, copy number analysis and identification of the source of an organism or sample, or the population from which an organism or sample originates. Any analysis of genomic DNA may be benefited by a reproducible method of polymorphism analysis. Furthermore, the probes, sequences, arrays and collections of SNPs of the presently claimed invention are particularly well suited for study and characterization of human genomic samples from individuals and populations.

In a preferred embodiment, the disclosed arrays, sequences and probes are used to genotype individuals, populations or samples. For example, any of the procedures described above, alone or in combination, could be used to interrogate SNPs in the collection. The disclosed arrays could be used in conjunction with methods of reducing the complexity of a sample in a reproducible and predictable manner. For example, complexity reduction methods may be designed to amplify a collection of target sequences that correspond to fragments containing SNPs from the sequence listing. Genotyping assays may be designed to interrogate the genotypes of each of the SNPs represented by a sequence in SEQ ID Nos. 1-10,204. The assay or assays may be used to genotype the SNPs in a plurality of individuals to determine allele frequency and association of alleles with particular phenotypes. Methods of use for polymorphisms and SNP discovery can be found in, for example, U.S. Pat. No. 6,361,947 which is herein incorporated by reference in its entirety for all purposes.

The disclosed collection of SNPs may be used for a variety of genetic analysis methods, for example, linkage studies, association studies, pharmacogenomics analysis, forensics, paternity testing, determination of relatedness, anthropological studies, and copy number analysis.

EXAMPLE

The following example serves to illustrate the type of experiment that could be conducted using the invention.

Polymorphism Detection by Hybridization to High Density Oligonucleotide Arrays

Arrays containing the desired number of probes can be synthesized using the method described in U.S. Pat. No. 5,143,854, incorporated by reference above. An exemplary array, the Mapping 10K 2.0 (Xba 142) array may be purchased from Affymetrix, Inc., Santa Clara.

Genomic DNA was digested with XbaI by mixing 5 μl 50 ng/μl human genomic DNA (Coriell Cell Repositories) with 10.5 μl H₂O (Accugene), 2 μl 10× RE buffer 2 (NEB, Beverly, Mass.), 2 μl 10×BSA (NEB, Beverly, Mass.), and 0.5 μl of 0.5 U/μl XbaI (NEB, Beverly, Mass.). The reaction was incubated at 30° C. for 2 hours, then the enzyme was inactivated by incubation at 70° C. for 20 min and then to 4° C. The reaction may be stored at −20° C. The reaction was performed in a 96 well plate and incubations were performed in a thermal cycler.

For ligation of the adapters the digested DNA was then mixed with 1.25 μl 5 μM adaptor in TE pH 8.0, 2.5 μl T4 DNA ligation buffer and 1.25 μl T4 DNA Ligase (NEB, Beverly, Mass.) which is added last. The reaction was incubated at 16° C. for 2 hours then at 70° C. for 20 min and then to 4° C. The 25 μl ligation mixture is then diluted with 75 μl H₂O and may be stored at −20° C.

For PCR 10 μl of the diluted ligated DNA is mixed with 10 μl PCR buffer II (Applied Biosystems), 10 μl (2.5 mM each) dNTP (PanVera Takara, Madison, Wis.), 10 μl 25 mM MgCl₂, 7.5 μl 10 μM primer (for a final concentration of 0.75 μM), 2 μl 5 U/μl AmpliTaq Gold® (Applied Biosystems) and 50.5 μl H₂O. For each array four 100 μl reactions were prepared. Amplification was done using the following program: 95° C. for 3 min; 35 cycles of 95° C. for 20 sec, 59° C. for 15 sec and 72° C. for 15 sec; and a final incubation at 72° C. for 7 min. The reactions were then held at 4° C. The lid heating option was selected.

The PCR reactions were then purified by mixing the 100 μl PCR reaction with 500 μl PB or PM buffer into Qiagen columns (Valencia, Calif.) and the column was centrifuged at 13,000 rpm for 1 min. Flow through was discarded and 750 μl PE buffer with ethanol was added into the column to wash the sample and the column was spun at 13,000 rpm for 1 min. The flow through was discarded and the column was spun at 13,000 rpm for another 1 min. The flow through was discarded and the column was placed in a new collection tube. For 2 of the 4 samples 30 μl of EB elution buffer pH 8.5 was added to the center of the QIAquick membrane to elute the sample and the columns were allowed to stand at room temperature for 5 min and then centrifuged at 13,000 for 1 min. The elution buffer from the first 2 samples was then used to elute the other 2 samples and the eluates were combined. The DNA was quantified and diluted so that 48 μl contains 20 μg DNA.

The DNA was fragmented by mixing 48 μl DNA (20 μg), 5 μl RE Buffer 4, and 2 μl 0.09 U/μl DNase in a total volume of 55 μl. The reaction was incubated at 37° C. for 30 min then 95° C. for 15 min and then held at 4° C.

Fragments were labeled by incubating 50 μl fragmented DNA, 13 μl 5× TdT buffer (Promega, Madison, Wis.), 1 μl 1 mM biotinolated-ddATP (NEN Life Sciences, Boston, Mass.), and 1 μl TdT (Promega, Madison, Wis.) at 37° C. overnight then at 95° C. for 10 min, then held at 4° C.

Hybridization mix is 12 μl 1.22 M MES, 13 μl DMSO, 13 μl 50× Denharts, 3 μl 0.5M EDTA, 3 μl 10 mg/ml herring sperm DNA, 3 μl 10 nM oligo B2, 3 μl 1 mg/ml Human Cot-1, 3 μl 1% Tween-20, and 140 μl 5M TMACL. 70 μl labeled DNA was mixed with 190 μl hybridization mix. The mixture was incubated at 95° C. for 10 min, spun briefly and held at 47.5° C. 200 μl of the denatured mixture was hybridized to an array at 47.5° C. for 16 to 18 hours at 60 rpm. The array comprised SEQ ID NOS. 1-10,204tiled on an array along with mismatch, antisense match and antisense mismatch probes for each of the sequences. Each probe is present in a spatially addressable location.

Staining mix was 990 μl H₂O, 450 μl 20×SSPE, 15 μl Tween-20, 30 μl 50% Denharts. For the first stain mix 495 μl staining mix with 5 μl 1 mg/ml streptavidin (Pierce Scientific, Rockford, Ill.), for the second stain mix 495 μl staining mix with 5 μl 0.5 mg/ml biotinylated anti-streptavidin antibody (Vector Labs, Burlingame, Calif.) and for the third stain mix 495 μl staining mix with 5 μl 1 mg/ml streptavidin, R-phycoerythrin conjugate (Molecular Probes, Eugene, Oreg.). Wash and stain under standard conditions.

Hybridized samples were analyzed with a computer system to determine which alleles were present for a particular SNP. See also, the GeneChip® Mapping 10K 2.0 Assay Manual, Rev 1, 2004.

To prepare multiple samples simultaneously the PCR reactions may be performed in multiwell plates. PCR purification and elution of reactions performed in 96 well plates may be performed using the QIAGEN Min Elute 96 UF PCR purification plate as follows. Connect a vacuum manifold to a vacuum source so that ˜800 mbar is maintained. Place a waste tray inside base of the manifold. Place a MinElute 96 UF PCR Purification Plate on top of the manifold. Cover wells that are not needed with PCR plate cover. Consolidate 4 PCR reactions for each sample into one well of the plate. Apply vacuum and maintain ˜800 mbar until the wells are completely dry, about 90 minutes for 400 μl sample. Wash the PCR products with 50 μl water and dry the wells completely. Repeat 2 times for a total of 3 water washes. Turn off vacuum and remove plate. Tap the plate to remove any liquid that may remain on the bottom of the plate. Add 40 μl EB buffer to each well and cover the plate with PCR plate cover film. Shake the plate for 5 minutes moderately. Recover the PCR product by pipetting the eluate out of each well.

Quantify and normalize PCR product and transfer 20 μg of each sample in 45 μl EB buffer to new plate for fragmentation. Add 5 μl 10× fragmentation buffer to each sample on ice and vortex for 2 seconds. Add 5 μl of diluted fragmentation reagent (0.48 U/μl) to each reaction. Vortex for 1 minute, incubate 30 min at 37° C., 15 min at 95° C. and hold at 4° C.

Mix 14 μl 5× TdT buffer, 2 μl GeneChip DNA Labeling Reagent (5 nM) and 3.4 μl TdT (30 U/μl) with 50.6 μl fragmented DNA reaction. Seal the plate with a plate cover, vortex 2 minutes, incubate at 37° C. for 2 hours, 95° C. for 15 minutes and hold at 4° C. Mix the labeled DNA with 190 μl Hybridization Mix and denature at 95° C. for 10 minutes. Inject 80 μl into the array and hybridize at 48° C. for 16 to 18 hours at 60 rpm. Wash, stain and scan according to the manufacturers instructions, see GeneChip Mapping 10K 2.0 Assay Manual.

CONCLUSION

The inventions herein provide a pool of unique nucleic acid sequences, which may be used to genotype a collection of Human SNPs. These sequences can be used for a variety of types of analyses.

The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. The scope of the invention should, therefore, be determined not with reference to the above description, but instead be determined with reference to the appended claims along with their full scope of equivalents. 

1. An array of oligonucleotides, the array comprising: at least 10,000 different sequence oligonucleotides attached to a solid support, wherein each different sequence oligonucleotide comprises at least 20 contiguous nucleotides of a different sequence listed in SEQ ID Nos. 1-10,204 and wherein each different sequence oligonucleotide is attached to a solid support in a determinable location of the array.
 2. The array of claim 1 wherein the array comprises at least 100,000 different sequence oligonucleotides.
 3. The array of claim 1 wherein the array comprises at least 10,204 different sequence oligonucleotides wherein each oligonucleotide comprises at least 20 contiguous nucleotides from a sequence listed in SEQ ID Nos. 1-10,204 and wherein each oligonucleotide comprises sequence from a different sequence listed in SEQ ID NOS. 1-10,204.
 4. The array of claim 1 wherein each different sequence oligonucleotide is attached to a bead.
 5. An array of oligonucleotide probes wherein the array comprises a probe set to interrogate each of the SNPs represented by SEQ ID Nos. 1-10,204, wherein a probe set comprises one or more probe quartets, wherein a probe quartet comprises a perfect match probe for each allele of each SNP and a mismatch probe for each allele of each SNP.
 6. The array of claim 5 wherein a probe set comprises a plurality of probe quartets for each SNP wherein each probe quartet in the plurality is shifted relative to other probe quartets in the plurality in the position of the polymorphic base.
 7. The array of claim 6 wherein the polymorphic base in each quartet in said plurality of quartets is in a position selected from −7, −6, −5, −4, −3, −2, −1, 0, 1, 2, 3, 4, 5, 6, or 7, wherein 0 is the central position of the probe.
 8. The array of claim 5 wherein each probe is attached to a different bead.
 9. A kit for genotyping at least 1,000 of the SNPs represented by SEQ ID Nos. 1-10,204, said kit comprising: a first plurality of oligonucleotides, wherein each oligonucleotide in the plurality of oligonucleotides comprises 15 to 33 bases of a sequence from SEQ ID Nos. 1-10,204 or the complement thereof and a second plurality of mismatch oligonucleotides corresponding to each oligonucleotide in said first plurality of oligonucleotides and wherein each different oligonucleotide is attached to a solid support in a determinable location.
 10. A kit comprising a first plurality of oligonucleotides wherein each oligonucleotide in said first plurality of oligonucleotides comprises at least 15 contiguous nucleotides from a sequence selected from SEQ ID Nos. 10,137-10,204 or the complements thereof and wherein each oligonucleotide in said first plurality of oligonucleotides is attached to a solid support.
 11. The kit of claim 10 wherein the solid support is a plurality of beads.
 12. The kit of claim 10 wherein the solid support is glass.
 13. The kit of claim 10 further comprising a second plurality of oligonucleotides wherein each oligonucleotide in said second plurality of oligonucleotides comprises at least 15 contiguous nucleotides from a sequence selected from SEQ ID Nos. 1-10,136 or the complements thereof and wherein each oligonucleotide in said second plurality of oligonucleotides is attached to a solid support. 